α-Pinene, a Main Component of Pinus Essential Oils, Enhances the Expression of Insulin-Sensitive Glucose Transporter Type 4 in Murine Skeletal Muscle Cells.

Glucose transporter-4 (GLUT4) represents the major glucose transporter isoform responsible for glucose uptake into insulin-sensitive cells, primarily in skeletal muscle and adipose tissues. In insulin-resistant conditions, such as type 2 diabetes mellitus, GLUT4 expression and/or translocation to the cell plasma membrane is reduced, compromising cell energy metabolism. Therefore, the use of synthetic or naturally occurring molecules able to stimulate GLUT4 expression represents a good tool for alternative treatments of insulin resistance. The present study aimed to investigate the effects of essential oils (EOs) derived from Pinus spp. (P. nigra and P. radiata) and of their main terpenoid constituents (α- and ß-pinene) on the expression/translocation of GLUT4 in myoblast C2C12 murine cells. For this purpose, the chemical profiles of the EOs were first analyzed through gas chromatography-mass spectrometry (GC-MS). Cell viability was assessed by MTT assay, and GLUT4 expression/translocation was evaluated through RT-qPCR and flow cytometry analyses. The results showed that only the P. nigra essential oil (PnEO) and α-pinene can increase the transcription of the Glut4/Scl2a4 gene, resulting in a subsequent increase in the amount of GLUT4 produced and its plasma membrane localization. Moreover, the PnEO or α-pinene can induce Glut4 expression both during myogenesis and in myotubes. In summary, the PnEO and α-pinene emulate insulin's effect on the GLUT4 transporter expression and its translocation to the muscle cell surface.

Characterization of Stable Pyrazole Derivatives of Curcumin with Improved Cytotoxicity on Osteosarcoma Cell Lines.

Curcumin (CUR) is a natural molecule that is unstable due to the presence of a bis-ketone. To obtain more stable derivatives in biological fluids, the bis-ketone was replaced with pyrazole or O-substituted oximes. Their stability in solution was studied by UV-visible spectrophotometry. The effects on proliferation were studied by MTT assay and/or clonogenicity assay. Induction of apoptosis was evaluated by annexin V staining and Western blot analysis. The bioavailability was obtained from the analysis of the molecular chemical-physical characteristics. The replacement of the bis-ketone with a pyrazole ring or O-substituted oximes improved the stability of all the CUR-derivative molecules. These derivatives were more stable than CUR in solution and were generally cytotoxic on a panel of cancer cell lines tested, and they promoted caspase-dependent apoptosis. Derivative 1 was the most potent in the osteosarcoma (OS) lines. With respect to CUR, this derivative showed cytotoxicity at least three times higher in the MTT assay. In addition, in the clonogenic assay, 1 maintained the activity in conditions of long treatment presumably by virtue of its improved stability in biological fluids. Notably, 1 should have improved chemical-physical characteristics of bioavailability with respect to CUR, which should allow for reaching higher blood levels than those observed in the CUR trials. In conclusion, 1 should be considered in future clinical studies on the treatment of OS, either alone or in combination with other medications currently in use.

Cytotoxicity of Isoxazole Curcumin Analogs on Chronic Myeloid Leukemia-Derived K562 Cell Lines Sensitive and Resistant to Imatinib.

Despite curcumin (CUR) inhibiting cell proliferation in vitro by activating apoptotic cell death, its use in pharmacological therapy is hampered by poor solubility, low stability in biological fluids, and rapid removal from the body. Therefore, CUR-derivatives with better biological and chemical-physical characteristics are needed. The bis-ketone moiety of CUR strongly influences its stability in slightly alkaline solutions such as plasma. Here, we considered its replacement with isoxazole, beta-enamine, or oxime groups to obtain more stable derivatives. The evaluation of the chemical-physical characteristics showed that only of the isoxazole derivatives 2 and 22 had better potential than CUR in terms of bioavailability. The UV-visible spectrum analysis showed that derivatives 2 and 22 had better stability than CUR in solutions mimicking the biological fluids. When tested on a panel of cell lines, derivatives 2 and 22 had marked cytotoxicity (IC50 = 0.5 µM) compared with CUR only (IC50 = 17 µM) in the chronic myeloid leukemia (CML)-derived K562 cell line. The derivative 22 was the more selective for CML cells. When administered at the average concentration found for CUR in the blood of patients, derivatives 2 and 22 had potent effects on cell cycle progression and apoptosis initiation, while CUR was ineffective. The apoptotic effect of derivatives 2 and 22 was associated with low necrosis. In addition, derivative 22 was able to reverse drug resistance in K562 cells resistant to imatinib (IM), the reference drug used in CML therapy. The cytotoxicity of derivative 22 on IM-sensitive and resistant cells was associated with upregulation of FOXN3 and CDKN1A expression, G2/M arrest, and triggering of apoptosis. In conclusion, derivative 22 has chemical-physical characteristics and biological effects superior to CUR, which allow us to hypothesize its future use in the therapy of CML and CML forms resistant to IM, either alone or in combination with this drug.

A Central Contribution of TG2 Activity to the Antiproliferative and Pro-Apoptotic Effects of Caffeic Acid in K562 Cells of Human Chronic Myeloid Leukemia.

Caffeic acid (CA) has shown antitumor activity in numerous solid and blood cancers. We have recently reported that CA is active in reducing proliferation and triggering apoptosis in both Imatinib-sensitive and resistant Chronic Myeloid Leukemia (CML) cells. Tissue transglutaminase type 2 (TG2) enzyme is involved in cell proliferation and apoptosis of numerous types of cancer. However, its activity has different effects depending on the type of tumor. This work investigated the possible involvement of TG2 activation in the triggering of CA-dependent anticancer effects on the K562 cell line, which was studied as a model of CML. CA-dependent changes in TG2 activity were compared with the effects on cell proliferation and apoptosis. The use of N-acetylcysteine (NAC), an antioxidant molecule, suggested that the antiproliferative effect of CA was due to the increase in reactive oxygen species (ROS). The use of a TG2 inhibitor showed that TG2 activity was responsible for the increase in ROS generated by CA and reduced both caspase activation and triggering of CA-dependent apoptosis. The knocking-down of TGM2 transcripts confirmed the crucial involvement of TG2 activation in CML cell death. In conclusion, the data reported, in addition to ascertaining the important role of TG2 activation in the antiproliferative and pro-apoptotic mechanism of CA allowed us to hypothesize a possible therapeutic utility of the molecules capable of triggering the activation pathways of TG2 in the treatment of CML.

Caffeic Acid Enhances the Anti-Leukemic Effect of Imatinib on Chronic Myeloid Leukemia Cells and Triggers Apoptosis in Cells Sensitive and Resistant to Imatinib.

Among the phenolic acids tested on the K562 cell line, a model of chronic myeloid leukemia (CML), caffeic acid (CA) was biologically active on sensitive and imatinib (IM)-resistant cells at micro-molar concentration, either in terms of reduction of cell proliferation or triggering of apoptosis. The CA treatment provoked mitochondrial membrane depolarization, genomic DNA fragmentation and phosphatidylserine exposure, hallmarks of apoptosis. Cell cycle analysis following the treatment with comparable cytotoxic concentrations of IM or CA showed marked differences in the distribution profiles. The reduction of cell proliferation by CA administration was associated with increased expression of two cell cycle repressor genes, CDKN1A and CHES1, while IM at a cytotoxic concentration increased the CHES1 but not the CDKN1A expression. In addition, CA treatment affected the proliferation and triggered the apoptosis in IM-resistant cells. Taken together, these data suggested that CA induced the anti-proliferative effect and triggered apoptosis of CML cells by a different mechanism than IM. Finally, the combined administration of IM and CA at suboptimal concentrations evidenced a synergy of action in determining the anti-proliferative effect and triggering apoptosis. The ability of CA to potentiate the anti-leukemic effect of IM highlighted the nutraceutical potential of CA in CML.

Selected terpenes from leaves of Ocimum basilicum L. induce hemoglobin accumulation in human K562 cells.

Re-expression of fetal hemoglobin (HbF) was proposed as a possible therapeutic strategy for ß-haemoglobinopathies. Although several inducers of HbF were tested in clinical trials, only hydroxyurea (HU) received FDA approval. Despite it produced adequate HbF levels only in half of HU-treated SCD patients, and was ineffective at all in ß-thalassemia patients, beneficial effects of this approach suggested to continue in this direction identifying further molecules capable of inducing HbF. We tested the potential of essential oil isolated from Ocimum basilicum L. leaves (ObEO) in inducing hemoglobin biosynthesis. Initially, dose-dependent effect and kinetics of hemoglobin accumulation in K562 cells after treatment with ObEO were evaluated. ObEO induced dose-dependent hemoglobin accumulation superior to hydroxyurea and rapamycin and a strongest γ-globin mRNA expression. Terpenes composition of ObEO was studied by GC-MS. Three main constituents, linalool, eugenol and eucalyptol, represented about 75% of total. A blend of these three terpenes fully replicated the ObEO's biological effect, thus indicating that one of them or all together could be the active ingredients. When terpenes were tested individually, eugenol was the only one inducing stable hemoglobin accumulation, while eucalyptol and linalool produced only a small transient response. However, eugenol potential was strongly enhanced in the presence of eucalyptol and linalool, suggesting a synergistic effect on hemoglobin accumulation. By these results, the discovery of a new inducer and the interesting activity of a blend of major terpenes from ObOE on Hb accumulation could have positive fallouts on ß-thalassemia and sickle cells anemia.

Chemical Composition of Essential Oils from Thymus vulgaris, Cymbopogon citratus, and Rosmarinus officinalis, and Their Effects on the HIV-1 Tat Protein Function.

New drugs would be beneficial to fight resistant HIV strains, in particular those capable of interfering with essential viral functions other than those targeted by highly active antiretroviral therapy drugs. Despite the central role played by Tat protein in HIV transcription, a search for vegetable extracts able to hamper this important viral function was never carried out. In this work, we evaluated the chemical composition and possible interference of essential oil from Thymus vulgaris, Cananga odorata, Cymbopogon citratus, and Rosmarinus officinalis with the Tat/TAR-RNA interaction and with Tat-induced HIV-1 LTR transcription. GC/MS Analysis demonstrated the biodiversity of herbal species translated into essential oils composed of different blends of terpenes. In all of them, 4 - 6 constituents represent from 81.63% to 95.19% of the total terpenes. Essential oils of Thymus vulgaris, Cymbopogon citratus, and Rosmarinus officinalis were active in interfering with Tat functions, encouraging further studies to identify single terpenes responsible for the antiviral activity. In view of the quite different composition of these essential oils, we concluded that their interference on Tat function depends on specific terpene or a characteristic blend.

Proposal for a novel management of indeterminate thyroid nodules on the basis of cytopathological subclasses.

Indeterminate thyroid nodules include heterogeneous lesions that could benefit from a differential management. Our aim is to better define the management of the Bethesda System for Reporting Thyroid Cytopathology class III and IV nodules, by identifying cytological subcategories among Bethesda System for Reporting Thyroid Cytopathology class III associated with different clinical risk, by means of ultrasound, repeated FNAB, and BRAFV600E molecular analysis. We also evaluated the outcome of nodules not operated, over a 5-year follow-up. Out of 460 nodules (269 Bethesda System for Reporting Thyroid Cytopathology class III and 191 Bethesda System for Reporting Thyroid Cytopathology class IV), 344 were operated on surgical group and 116 followed-up conservatively (follow-up group). Bethesda System for Reporting Thyroid Cytopathology class III was divided into four subcategories on the basis of cytomorphological features (III-1, III-2, III-3, III-4). Clinical risk was defined on the basis of histological, cytological, and ultrasound data. Malignancy was higher in Bethesda System for Reporting Thyroid Cytopathology class III vs. Bethesda System for Reporting Thyroid Cytopathology class IV (34.4 vs. 26.2 %; p < 0.01). Papillary thyroid carcinoma was the most frequent cancer in each Bethesda System for Reporting Thyroid Cytopathology class (35 %). BRAFV600E diagnostic accuracy was 87 %. Repeated FNAB reclassified as benign nearly 40 % of nodules, selecting patients where surgery could be spared. Significant nodule growth occurred in 13.7 % of nodules, belonging mostly to Bethesda System for Reporting Thyroid Cytopathology class III-2 and Bethesda System for Reporting Thyroid Cytopathology class IV. Overall clinical risk was higher in Bethesda System for Reporting Thyroid Cytopathology III-1, III-4, and IV classes. We propose a differential management of Bethesda System for Reporting Thyroid Cytopathology III and IV classes and related subcategories: surgery may be indicated in Bethesda System for Reporting Thyroid Cytopathology class III-1, III-4, and IV; a conservative follow-up avoiding repeated FNAB may be appropriated in class III-3, while repeated FNAB may be useful in class III-2.

Growth Hormone differentially modulates chemoresistance in human endometrial adenocarcinoma cell lines.

Growth Hormone may influence neoplastic development of endometrial epithelium towards endometrial adenocarcinoma, which is one of the most occurring tumors in acromegalic patients. Since chemoresistance often develops in advanced endometrial adenocarcinoma, we investigated whether Growth Hormone might influence the development of chemoresistance to drugs routinely employed in endometrial adenocarcinoma treatment, such as Doxorubicin, Cisplatin, and Paclitaxel. Growth Hormone and Growth Hormone receptor expression was assessed by immunofluorescence in two endometrial adenocarcinoma cell lines, AN3 CA and HEC-1-A cells. Growth Hormone effects were assessed investigating cell viability, caspase3/7 activation, ERK1/2, and protein kinase C delta protein expression. AN3 CA and HEC-1-A cells display Growth Hormone and Growth Hormone receptor. Growth Hormone does not influence cell viability in both cells lines, but significantly reduces caspase 3/7 activation in AN3 CA cells, an effect blocked by a Growth Hormone receptor antagonist. Growth Hormone rescues AN3 CA cells from the inhibitory effects of Doxorubicin and Cisplatin on cell viability, while it has no effect on Paclitaxel. Growth Hormone does not influence the pro-apoptotic effects of Doxorubicin, but is capable of rescuing AN3 CA cells from the pro-apoptotic effects of Cisplatin. On the other hand, Growth Hormone did not influence the effects of Doxorubicin and Paclitaxel on HEC-1A cell viability. The protective action of Growth Hormone towards the effects of Doxorubicin may be mediated by ERK1/2 activation, while the pro-apoptotic effects of Cisplatin may be mediated by protein kinase C delta inhibition. All together our results indicate that Growth Hormone may differentially contribute to endometrial adenocarcinoma chemoresistance. This may provide new insights on novel therapies against endometrial adenocarcinoma chemoresistant aggressive tumors.

Protein Kinase C Delta restrains growth in ACTH-secreting pituitary adenoma cells.

Protein Kinase C Delta (PRKCD) has been highlighted among disrupted pathways in corticotroph adenomas. PRKCD is expressed at low level in human corticotroph adenomas and controls cell cycle in vitro. Therefore, PRKCD may play an important role in the development/progression of corticotroph adenomas, warranting further studies to understand the role of PRKCD and related pathways in restraining pituitary cell growth. We evaluated PRKCD role in influencing cell behavior in terms of cell viability, hormone expression and protein expression profile, by silencing PRKCD in AtT-20/D16v-F2 cells. PRKCD silencing increases cell viability, enhances hormone expression and induces morphological changes associated with deregulation of adhesion molecules. PRKCD silencing is associated with an increase in Epithelial Growth Factor Receptor (EGFR) expression, a marker of tumor aggressive behavior, and sensitivity to anti-EGFR molecules. PRKCD might restrain corticotroph adenoma cells from acquiring an aggressive behavior, candidating PRKCD as a possible molecular target for the treatment of corticotroph adenomas.

Inhibition of epithelial growth factor receptor can play an important role in reducing cell growth and survival in adrenocortical tumors.

Medical treatment of adrenocortical carcinoma (ACC) is still far from optimal, since even molecular targeted therapy failed to demonstrate striking results. Clinical trials enrolling ACC patients with high tissue vascular endothelial growth factor receptor (VEGFR) expression levels showed controversial results after treatment with Sunitinib, possibly due to variability in the expression of drug targets, which include epidermal growth factor receptor (EGFR). To better clarify this issue, we evaluated whether VEGFR may play a crucial role in ACC responsiveness to Sunitinib and whether EGFR may represent an alternative target in ACC medical treatment, by employing two ACC cell lines, the NCI-H295 and SW13 cells lines, and adrenocortical tissues primary cultures. Our data show that VEGF/VEGFR system may not be crucial in modulating ACC proliferation and responsiveness to Sunitinib. In addition, by cell viability, proliferation and caspase activation assays we found that Sunitinib inhibits adrenocortical cell viability acting, at least in part, through EGFR, that, in turn, is crucial for EGF proliferative effect on adrenocortical cells. The latter depends, at least in part, on ERK 1/2 activation. An EGFR selective inhibitor was highly effective in reducing cell viability in an adrenocortical tumor primary culture and in the SW13 cells, which express high EGFR levels. Our results suggest that EGFR inhibitors could represent effective therapeutic tools in ACC patients whose tumors express high EGFR levels, that, in turn, may be considered a predictive factor of response. Accurate molecular tumor profiling is crucial to predict drug efficacy and to tailor ACC patients therapeutic approach.

Igf-I influences everolimus activity in medullary thyroid carcinoma.

CONTEXT: Medullary thyroid carcinoma (MTC) is a rare tumor originating from thyroid parafollicular C cells. It has been previously demonstrated that insulin-like growth factor I (IGF-I) protects MTC from the effects of antiproliferative drugs. Everolimus, an mTOR inhibitor, has shown potent antiproliferative effects in a human MTC cell line, TT, and in two human MTC primary cultures. OBJECTIVE: To verify whether IGF-I may influence the effects of everolimus in a group of human MTC primary cultures. DESIGN: We collected 18 MTCs that were dispersed in primary cultures, treated without or with 10 nM-1 µM everolimus and/or 50 nM IGF-I. Cell viability was evaluated after 48 h, and calcitonin (CT) secretion was assessed after a 6 h incubation. IGF-I receptor downstream signaling protein expression profile was also investigated. RESULTS: Everolimus significantly reduced cell viability in eight MTC [by ~20%; P < 0.01 vs. control; everolimus-responders (E-R) MTCs], while cell viability did not change in 10 MTCs [everolimus-non-responders (E-NR) MTCs]. In E-R MTCs, IGF-I blocked the antiproliferative effects of everolimus that did not affect CT secretion, but blocked the stimulatory effects of IGF-I on this parameter. IGF-I receptor downstream signaling proteins were expressed at higher levels in E-NR MTC as compared to E-R MTCs. CONCLUSION: IGF-I protects a subset of MTC primary cultures from the antiproliferative effects of everolimus and stimulates CT secretion by an mTOR mediated pathway that, in turn, may represent a therapeutic target in the treatment of aggressive MTCs.

The expression of the truncated isoform of somatostatin receptor subtype 5 associates with aggressiveness in medullary thyroid carcinoma cells.

The truncated somatostatin receptor variant sst5TMD4 associates with increased invasiveness and aggressiveness in breast cancer. We previously found that sst5 activation may counteract sst2 selective agonist effects in a medullary thyroid carcinoma (MTC) cell line, the TT cells, and that sst5TMD4 is overexpressed in poorly differentiated thyroid cancers. The purpose of this study is to evaluate sst5TMD4 expression in a series of human MTC and to explore the functional role of sst5TMD4 in TT cells. We evaluated sst5TMD4 and sst5 expression in 36 MTC samples. Moreover, we investigated the role of sst5TMD4 in TT cells evaluating cell number, DNA synthesis, free cytosolic calcium concentration ([Ca(2+)]i), calcitonin and vascular endothelial growth factor levels, cell morphology, protein expression, and invasion. We found that in MTC the balance between sst5TMD4 and sst5 expression influences disease stage. sst5TMD4 overexpression in TT cells confers a greater growth capacity, blocks sst2 agonist-induced antiproliferative effects, modifies the cell phenotype, decreases E-cadherin and phosphorylated ß-catenin levels, increases vimentin, total ß-catenin and phosphorylated GSK3B levels (in keeping with the development of epithelial to mesenchymal transition), and confers a greater invasion capacity. This is the first evidence indicating that sst5TMD4 is expressed in human MTC cells, where it associates with more aggressive behavior, suggesting that sst5TMD4 might play a functionally relevant role.

Early onset acromegaly associated with a novel deletion in CDKN1B 5'UTR region.

Genetic alterations frequently are involved in the development of a pituitary adenoma in young age. We here characterize the functional role of a deletion in CDKN1B 5'-UTR region (c.-29_-26delAGAG) identified in an acromegalic patient that developed a growth hormone in pituitary adenoma during childhood. Our results show that the identified novel heterozygous deletion in the CDKN1B 5'-UTR region associates with a reduction in CDKN1B mRNA levels, a predicted altered secondary mRNA structure, and a reduced CDKN1B 5'-UTR transcriptional activity in vitro. The patient displayed loss of heterozygosity in the same CDKN1B 5'-UTR region at tissue level and the 5'UTR region containing the deleted sequence encompasses a GRE. These findings indicate that the identification of functional alterations of newly discovered genetic derangements need to be fully characterized and always correlated with the clinical manifestations.

Relevance of BRAF(V600E) mutation testing versus RAS point mutations and RET/PTC rearrangements evaluation in the diagnosis of thyroid cancer.

BACKGROUND: A molecular profile including BRAF and RAS mutations as well as RET/PTC rearrangement evaluation has been proposed to provide an accurate presurgical assessment of thyroid nodules and to reduce the number of unnecessary diagnostic surgeries, sparing patients' health and saving healthcare resources. However, the application of such molecular analyses may provide different results among different centers and populations in real-life settings. Our aims were to evaluate the diagnostic utility of assessing the presence of BRAF and RAS mutations and RET/PTC1 and RET/PTC3 rearrangements in all cytological categories in an Italian group of thyroid nodule patients assessed prospectively, and to understand whether and which mutation testing might be helpful in cytologically indeterminate nodules. METHODS: A total of 911 patients were submitted to ultrasound and fine-needle aspiration biopsy examination. Cytological evaluation was performed in parallel with molecular testing and compared to pathological results in 940 thyroid nodules, including 140 indeterminate lesions. RESULTS: BRAF mutation testing provided the best contribution to cancer diagnosis, allowing the disease to be detected at an early stage, and identifying indeterminate nodules in which diagnostic lobectomy could be spared. On the contrary, RAS and RET/PTC analysis did not further increase diagnostic sensitivity for thyroid cancer. In addition, we found RET/PTC rearrangements in benign lesions, indicating that this molecular marker might not be useful for the detection of thyroid cancer. CONCLUSION: BRAF(V600E) mutation analysis is superior to RAS point mutations and evaluation of RET/PTC rearrangements in the diagnosis of thyroid cancer, even in indeterminate lesions.

Mitotane enhances doxorubicin cytotoxic activity by inhibiting P-gp in human adrenocortical carcinoma cells.

Mitotane is currently employed as adjuvant therapy as well as in the medical treatment of adrenocortical carcinoma (ACC), alone or in combination with chemotherapeutic agents. It was previously demonstrated that mitotane potentiates chemotherapeutic drugs cytotoxicity in cancer cells displaying chemoresistance due to P-glycoprotein (P-gp), an efflux pump involved in cancer multidrug resistance. The majority of ACC expresses high levels of P-gp and is highly chemoresistent. The aim of our study was to explore in vitro whether mitotane, at concentrations lower than those currently reached in vivo, may sensitize ACC cells to the cytotoxic effects of doxorubicin and whether this effect is due to a direct action on P-gp. NCI-H295 and SW13 cell lines as well as 4 adrenocortical neoplasia primary cultures were treated with mitotane and doxorubicin, and cell viability was measured by MTT assay. P-gp activity was measured by calcein and P-gp-Glo assays. P-gp expression was evaluated by Western blot. We found that very low mitotane concentrations sensitize ACC cells to the cytotoxic effects of doxorubicin, depending on P-gp expression. In addition, mitotane directly inhibits P-gp detoxifying function, allowing doxorubicin cytotoxic activity. These data provide the basis for the greater efficacy of combination therapy (mitotane plus chemotherapeutic drugs) on ACC patients. Shedding light on mitotane mechanisms of action could result in an improved design of drug therapy for patients with ACC.

N-carbamidoyl-4-((3-ethyl-2,4,4-trimethylcyclohexyl)methyl)benzamide enhances staurosporine cytotoxic effects likely inhibiting the protective action of Magmas toward cell apoptosis.

We recently demonstrated that Magmas overexpression protects GH-secreting rat pitutitary adenoma cell lines from apoptosis by inhibiting cytochrome c release from mitochondria after treatment with staurosporine, strongly suggesting a role of Magmas in preventing apoptosis. The aim of this study was to produce a drug that, by inhibiting Tim16, may sensitize chemoresistant tumor cell to proapoptotic stimuli. We synthesized six compounds and challenged their sensitizing effects toward the proapoptotic effects of staurosporine in the TT cell line, derived from a human medullary thyroid carcinoma. We found that compound 5, devoid of the planarity in the aliphatic part of the lead 1, is not cytotoxic but enhances the proapoptotic effects of staurosporine by reducing MMP activation. Compound 5 may be useful for cancer treatment in association with chemotherapeutic drugs, possibly allowing a reduction of the chemotherapeutic agent effective dose.

Deletion of exons 1-3 of the MEN1 gene in a large Italian family causes the loss of menin expression.

Multiple endocrine neoplasia type 1 (MEN1) syndrome is an autosomal dominant disease, characterized by parathyroid adenomas, endocrine gastroenteropancreatic tumors and pituitary adenomas, due to inactivating mutations of the MEN1 gene (chromosome 11q13). MEN1 mutations are mainly represented by nonsense, deletions/insertions, splice site or missense mutations that can be detected by direct sequencing of genomic DNA. However, MEN1 patients with large heterozygous deletions may escape classical genetic screening and may be misidentified as phenocopies, thereby hindering proper clinical surveillance. We employed a real-time polymerase chain reaction application, the TaqMan copy number variation assay, to evaluate a family in which we failed to identify an MEN1 mutation by direct sequencing, despite a clear clinical diagnosis of MEN1 syndrome. Using the TaqMan copy number variation assay we identified a large deletion of the MEN1 gene involving exons 1 and 2, in three affected family members, but not in the other nine family members that were to date clinically unaffected. The same genetic alteration was not found in a group of ten unaffected subjects, without family history of endocrine tumors. The MEN1 deletion was further confirmed by multiplex ligation-dependent probe amplification, which showed the deletion extended from exon 1 to exon 3. This new approach allowed us to correctly genetically diagnose three clinical MEN1 patients that were previously considered as MEN1 phenocopies. More importantly, we excluded the presence of genetic alterations in the unaffected family members. These results underline the importance of using a variety of available biotechnology approaches when pursuing a genetic diagnosis in a clinically suggestive setting of inherited endocrine cancer.

Correction: Magmas Overexpression Inhibits Staurosporine Induced Apoptosis in Rat Pituitary Adenoma Cell Lines.

[This corrects the article on p. e75194 in vol. 8.].

Magmas overexpression inhibits staurosporine induced apoptosis in rat pituitary adenoma cell lines.

Magmas is a nuclear gene that encodes for the mitochondrial import inner membrane translocase subunit Tim16. Magmas is overexpressed in the majority of human pituitary adenomas and in a mouse ACTH-secreting pituitary adenoma cell line. Here we report that Magmas is highly expressed in two out of four rat pituitary adenoma cell lines and its expression levels inversely correlate to the extent of cellular response to staurosporine in terms of apoptosis activation and cell viability. Magmas over-expression in rat GH/PRL-secreting pituitary adenoma GH4C1 cells leads to an increase in cell viability and to a reduction in staurosporine-induced apoptosis and DNA fragmentation, in parallel with the increase in Magmas protein expression. These results indicate that Magmas plays a pivotal role in response to pro-apoptotic stimuli and confirm and extend the finding that Magmas protects pituitary cells from staurosporine-induced apoptosis, suggesting its possible involvement in pituitary adenoma development.